We have continued our characterization of a putative neutralization escape mutant of HBV that emerged in individuals who were vaccinated against hepatitis B. Two licensed vaccines were investigated for their ability to protect chimpanzees against challenge with the S gene mutant. Both recombinant vaccines provided protection against challenge with the mutant virus, suggesting that properly vaccinated individuals are not at risk of infection by the S gene mutant virus. A bi-directional promoter complex was identified within the X gene of HBV, further emphasizing the complex genetic organization of this virus. Hepatitis B virus is being used as a model system for evaluating the new naked nucleic acid approach to vaccine development. Results obtained with various recombinant vectors and immunization protocols are being compared with each other and with extensive experience with other approaches to hepatitis B vaccine development that have been studied previously in the laboratory. Two chimpanzees were injected with 2 mg or 400 ug of plasmid DNA encoding the major and middle HBV envelope proteins. The animal receiving the larger dose of DNA attained antibody titers to HBV that were comparable to titers achieved following vaccination with commercial hepatitis B vaccine. The animal receiving the lower dose of DNA responded serologically but the antibody was of lower titer and transient. However, following vaccination with commercial hepatitis B vaccine 1 year later, both chimpanzees had an amnestic response to HBV, indicating that immunological memory had been achieved.